Pre-fermented symbiotic matrix based on a cereal suspension with encapsulated probiotics, manufacture process and corresponding utilization

ABSTRACT

The present invention is about a pre-fermented symbiotic matrix based on a cereal suspension, preferably oat, containing encapsulated probiotics and prebiotics, the manufacturing process and the corresponding utilization. The present invention&#39;s object is the development of a cereal symbiotic matrix, preferably from oat, pre-fermented with encapsulated probiotics and free and/or encapsulated prebiotics, with the aim of complementing the actual functional food market and solving problems inherent to the reduced shelf-life period of such foods due to loss of probiotic viability to values below the minimum limits needed in order to promote biological activity. Furthermore, the present invention&#39;s object is to improve the fermentative process conditions at different levels, namely fermentation time reduction in order to reduce energy consumption during the process and the risk of contamination reduction as well as promote long term microbial stability maintenance. The pre-fermented symbiotic matrix is designed, in particular, for those cases where intolerance and/or allergy to dairy products occur, yet it is further applicable to the pharmaceutical, cosmetic and preferably food industries, including pet food.

INVENTION FIELD

The present invention is related with the manufacture of a pre-fermentedsymbiotic matrix, containing prebiotics and/or probiotics, and it isapplicable to the pharmaceutical, cosmetic and preferably foodindustries, including pet food. The pre-fermented symbiotic matrix, freeor not of dairy ingredients, applies to all populations, in particularto those elements with intolerance and/or allergy to dairy products.

SUMMARY

The present invention's object is the development of a cereal symbioticmatrix, oat preferably, pre-fermented with encapsulated probiotics andfree and/or encapsulated prebiotics, with the purpose of complementingthe actual functional food market and solving problems inherent to thereduced shelf-life period of these foods. Moreover, the presentinvention's object is to improve the fermentative process conditions atdifferent levels, namely reduction of fermentation time as a means toeconomize energy during the manufacturing process, to reduce the risk ofcontamination and to maintain long term microbial stability.

INVENTION STATE OF THE ART

Over the last few decades, detailed knowledge on the influence of dieton human health has increased greatly, and populations across the worldhave become conscious of the need for a so-called ‘healthy diet’,justified by the life expectancy increases as well as representing animportant public health issue. With the increasing popularity ofprobiotic products among consumers, food companies need to face the callfor the manufacture of such products in order to appropriately meetconstant market requests. All food is functional, in the general meaningof the term, insofar as they supply energy and nutrients necessarytoward growth and maintenance. A food ingredient is considered asfunctional if it has been clearly demonstrated and scientificallyvalidated, in an efficient scientific way that it beneficially affectshealth, beyond the classical nutritional effect associated therewith.

This market is characterized by being dynamic and innovative with amarket quota of 10 to 15% and a growth rate of 20 to 30% per year, atworld level.

Probiotics can be defined as viable microorganisms that affect the hostbeneficially in as much as they promote the balance of its intestinalbacterial ecosystem. Lactobacillus, Bifidobacterium and Enterococcus,genera considered potentially probiotic, offer a protection to the hostagainst infections, considering that they prevent the attack, setting,response and/or virulence of specific enteropathogenics (antimicrobialactivity). These probiotics also have a beneficial effect in the controlof diarrhoeas, as well as in the reduction of the risk of development ofsome forms of cancer (anticarcinogenic activity). An effect on reductionlevels of blood cholesterol (hypocholesterolaemic activity) is alsodescribed. Another possible effect, which has been scientificallyvalidated, is the effect on the digestion of lactose, through theproduction of lactase (β-galactosidase) which facilitates the digestionof this sugar, and offers solutions for individuals intolerant tolactose. The probiotic products' beneficial effect is secured when thesecontain a minimum of 10⁶ CFU/ml, which is in agreement with theassumption of a minimum therapeutic dose per day suggested to be 10⁸ to10⁹ viable cells, which may be realisable through an intake ofapproximately 100 grams of product containing 10⁶-10⁷ viable cells permillilitre or gram. Probiotics present natural limitations to theirhealth benefits, due to their susceptibility to certain technologicaland functional factors, for instance high levels of oxygen, acidenvironments, freezing and the passage through the gastrointestinaltract.

Methods of encapsulation have begun to be applied, as a means toincrease the survival rate of probiotics, through their protection fromabovementioned adverse conditions. Microencapsulation is the technologyof packing solids, liquids or gases in very small capsules, capable ofreleasing their content at controlled rates and under specificconditions.

Several microencapsulating techniques are available, viz. emulsion andspray-drying. Emulsion encapsulation consists on adding a small volumeof solution containing microbial cells and polymers (discontinuousphase) to a greater volume of vegetable oil (continuous phase). Thismixture is then homogenized forming a water-in-oil emulsion. Onceobtained, the water soluble polymer must be insolubilized via a salinesolution with the objective of creating small gel particles in the oilphase. The size of the capsules can be controlled by varying the type ofstirring and its speed as well as the saline solution additionmechanism. The process of emulsion encapsulation is easily scaled-up andleads great survival rates of microorganisms (80 to 95%). The resultingcapsules present various sizes, which range from 25 μm to 50 μm.

On the other hand, the spray-drying method consists on drying an aqueousencapsulating agent mixture with viable microbial cells, using anatomizer. The drying occurs when the solution, after being vaporized,comes in contact with a hot air flow (entry temperature), and issubsequently, with the aid of a vacuum, gathered in the appropriaterecipient. This technology has as greatest advantages the low cost ofthe procedure, the easiness of the operation, the possibility of usingthermo-sensitive functional ingredients, the high quality/stability ofthe capsules obtained and the easy production in large quantities. Theobtained capsules can vary in size between 5 and 75 μm.

A prebiotic is by definition a non-digestible food ingredient whichpositively affects the host, stimulating selectively the growth and/oractivity of one or a limited number of bacteria in the colon.

The term symbiotic refers to a synergistic association of pre- andprobiotic agents with physiological activity in the same food.

Presently, prebiotics and their combination with probiotics in anencapsulated form consist in an investment for food industry, with theintention of maintaining their long term stability and optimizing thenutritional qualities of the associated product.

The document US2001/0016220 lists components of food products, whichcontain biological active ingredients that may be encapsulated, as wellas the process for their production and utilization. The componentsmentioned in this document comprise plant fibres including the onesproceeding from oats, soluble and insoluble polysaccharides, pectins,lenhins and gums. The biologically active components in the mentionedplant fibres may be probiotic microorganisms, prebiotics, enzymes,nutrients, secondary metabolites, natural or synthetic, substances withantioxidant activity, etc. The substances of encapsulation may beconstituted by polysaccharides (of plant or microbial origin),emulsifiers, peptides, proteins and prebiotic substances.

In terms of the process of attainment of these components, the documentin question foresees the introduction of biological active componentsinto an environment that contains substances that form the capsules.Even though this document describes products based on cereals containingbiologically active components, such as probiotics and/or prebiotics,encapsulated in a matrix formed by the plant fibres of the cereals, itdiffers considerably from the present invention, since this describes anaqueous suspension of pre-fermented cereals, with posteriorencapsulation of microorganisms, by emulsion, fluidized bed, or bydrying and subsequent addition of prebiotic components. In other words,in the previous document, all the components, prebiotics or probiotics,are encapsulated and are introduced in the matrix at the same phase,while this invention describes a process of attainment of thesecomponents in several phases, with protection of microbial activitythrough the encapsulation of microorganisms.

The document WO2005.002367 reports products and therapeuticalcompositions made of oats free of probiotic microorganisms, includingproteins, hydrolysed proteins and emulsifying lipids. Besides that, theproducts and compositions may still include β-glucans, and plantsterols. The corresponding production process is carried out via anenzymatic treatment of the oats fraction for removal of thecarbohydrates (preferentially by hydrolysis).

The document Wo02.065855 mentions non dairy products, made of cerealdispersions, containing β-glucans, proteins, natural sugars andproteins. The process to obtain such products uses enzymes, particularlyhydrolases, as well as isomerases, applied to cereal suspensions.

The document WO02/37984 reports products leavened by microbial culturesbased on oat suspensions, free of soy and milk, as well as thecorresponding manufacture. This document foresees the use ofLactobacillus and Streptococcus strains in the fermentation of the oatsuspension, as well as the inclusion of several components, such ascalcium hydrogeno-phosphate and/or calcium phosphate, β-glucan, maltose,maltodextrin, proteins, etc in an aqueous oat suspension which is laterincubated for fermentation.

The document WO00/65930 reports products made of cereals, particularlyoats, for further utilization as raw material in the food industry. Theprocess of attainment of these products includes the preparation of asuspension, from bran, flakes or flour of cereals. This suspension islater homogenized, at a predetermined temperature and pressure, in orderto obtain an emulsion. Afterwards, the emulsion can be leavened bymicroorganisms, such as Lactobacillus and Bifidobacterium, among others,acidified and finally pasteurized, (or even presented as a powder).

The document CA2383021 describes symbiotic compositions departing fromβ-glucans produced from cereals, obtained from flours or extracts ofcereals, inoculated by bacteria for fermentation. Lactobacillus,Streptococcus and/or Bifidobacterium are inoculated in aqueoussuspensions of cereals, treated with α-amylases, and added with astabilizing agent.

The document WO2004/037191 describes symbiotic products, in liquid orfrozen form, derived from soy or dairy products, composed of a mixtureof probiotic components (e.g. Lactobacillus and Bifidobacterium) andprebiotics, in which these may be constituted by polymers, particularly,inulin or oligofructose. The process of manufacture thereof uses amixture of prebiotic and probiotic components in a liquid phase;fermentation of this mixture occurs until pH reaches 4.5; and finalblend leads to the final product. At this final stage, it is, stillpossible to include a percentage of carbon dioxide.

The content described in the six aforementioned documents, differssubstantially from the content of our invention, since they describe thefermentation of oat suspensions, with the addition of freemicroorganisms or enzymes, in just one phase, and may eventually includeother additional components, while the invention under analysisdescribes suspensions of cereals pre-fermented by immobilizedmicroorganisms, to which encapsulated probiotics and free orencapsulated prebiotics are subsequently added.

When the microorganisms are used in the free form the shelf-life of thefinal product is reduced and the stability/viability of microorganismsover the storage period as well as in their passage through thegastro-intestinal tract is diminished, in comparison to the symbioticpre-fermented matrix with encapsulated probiotics (increase of 40 to60%), object of the present invention.

This present solution also solves the problems associated with reducedshelf-life mentioned in patents WO02.37984, WO00.65930, CA2383021,WO2004.037191 (an increase of 40 to 60% compared to the existingproducts with free microorganisms is reported) and maintains long termmicrobial stability.

The document EP 0 862 863 A2 has for object of invention the developmentof dried extruded cereals with surface and/or enclosed microorganisms,and with soluble fibre sources listing as examples of applicationbreakfast cereals and animal feed. The object of invention foresees thedevelopment of a cereal symbiotic matrix, preferentially in oatmeal,pre-fermented with encapsulated probiotics and free and/or encapsulatedprebiotics, which, when applied together, will confer a stabilizingeffect on the microorganisms present in the final product and favour thepassage through the gastrointestinal tract. Furthermore, an additionalobject of this invention is the health claim of cholesterol reductionassociated to p-glucan, as a source of non-digestible prebiotic solublefibres. The cereal suspension, preferentially oatmeal, is presented infresh, lyophilized, and frozen forms, adapted to the needs of theintervening parts in the food chain, and hence with several applicationsin the food industry.

The document WO2004.070026 discloses continuous processes concerningyeast immobilization in κ-carrageenan or alginate gel spheres, e.g. inbeer production, through formation of an emulsion e.g. with thecontinuous non-aqueous phase (plant oil) and the disperse aqueous phase(inoculated κ-carrageenan with yeast), using static stirrers. Thissubject differs from that disclosed in the present invention, becausethe immobilization process described, although pertaining to an emulsionbetween a plant oil and a microorganism-inoculated polymer, encompassesa yeast, whereas those in the present invention are all of probioticmicroorganisms.

The conceptualization of a phased process, to obtain symbiotic products,from pre-fermented cereal suspensions with added encapsulatedprobiotics, and subsequent incorporation of prebiotic compounds in thecereal matrix leads to a superior product, not only from a nutritionalpoint of view, due to long term microbiological stability maintenance,but also in what concerns.

GENERAL DESCRIPTION OF INVENTION

The present invention reports a cereal symbiotic matrix, preferentiallyoatmeal, pre-fermented with encapsulated probiotic and prebioticcompounds, its process of manufacture and its use in severalapplications, especially in the food industry but also in thepharmaceutical industry or similar counterparts.

The products obtained possess organoleptic characteristics that areidentical to those produced by traditional fermentation processes.

When encapsulated microorganisms are included, these products also havethe advantage of increasing their viability/stability, either as a longshelf-life or during passage through the gastro-intestinal tractfollowing ingestion.

The matrices also present, as an additional advantage, an extendedexpiration date up to 40% to 60% higher than those presented byavailable products on the market.

Through the use of this technology one obtains a pre-fermented productwith residual quantities of free microorganisms and with the sameorganoleptic characteristics as those of a traditionally fermentedproduct, being therefore a more valued product.

The immobilization technique of microbial cells confers advantages incomparison to free cell systems, such as: (i) reduction in fermentationtime up to 50 to 60%; (ii) increase of the microbial metabolism andstability; (iii) reduced risk of contamination; (iv) higher celldensity; (v) stable product quality associated with a decrease ofpost-acidification risk due to probiotic action, for example; (vi)improved substrate use and (vii) long time cell reutilization due toconstant cellular regeneration.

The process of obtaining these products reveals a method for improvementof the fermentative process conditions at several levels such as, (i)continuous reutilization of the immobilized cells; (ii) fermentationtime reduction contributes to energy saving throughout the process,(iii) reduction of contaminating risks and (iv) long term maintenance ofmicrobial stability.

DETAILED INVENTION DESCRIPTION 1. Preparation Process of OatmealSuspension

-   -   1.1. preparation of an oatmeal concentrate 5-20% (wiw) from        flakes, bran and/or flour and subsequent mixture in water;    -   1.2. heating of this mixture for 5 to 20 minutes in a        temperature range of 80 to 110° C., with continuous stirring;    -   1.3. grinding of the resulting preparation;    -   1.4. filtration of the obtained suspension; and    -   1.5. cooling of the mixture until a range of temperatures        between 25 and 48° C.

2. Process of Pre-Fermentation in Fluidized Bed Reactor Associated withCell Encapsulation by Emulsion

-   -   2.1. Process of pre-fermentation

The process of pre-fermentation is performed in a fluidized bed reactorwith immobilized microorganisms by cells obtained in steps 2.1.1.through 2.4.3.

The capsules are introduced in a column, with porosity smaller than thediameter of the capsules to induce the microorganisms-matrixinteraction, inside the pressurized reactor with constant and controlledbi-directional nitrogen flow. The immobilized cells inside the columnare reutilized in the fermentation process until they lose theirmetabolic properties.

-   -   2.1.1. Cell Culture Preparation    -   2.1.1.1. Preparation of the inocula from frozen cultures and        consequent activation by two consecutive transfers in MRS Broth        supplemented with L-cysteine-HCl 0.05% (w/v).    -   2.1.1.2. Inoculation of 1 to 20% (v/v) in 1000 mL of MRS Broth        (Man Rogosa and Sharpe) supplemented with L-cysteine-HCl 0.05%        (m/v) and subsequent incubation for 24 h at 37° C., under        anaerobic conditions, for Lactobacillus acidophilus Ki and 48 h        at 37° C. under anaerobic conditions for Bifidobacterium        animalis Bo and Bb12, for example.    -   2.1.1.3. Centrifugation of the resulting cultures at 4000 rpm        for 15 minutes, at 4° C., subsequent washing of pellet with, for        example, NaCl 0.9% (w/v) solution, and resuspension in 100 mL of        the same solution.    -   2.2. Polymer Solution Preparation    -   2.2.1. Preparation of a polymer solution, for example,        k-carrageenan 1 to 5% (w/v), with continuous stirring, variable        duration between 1 to 4 hours, temperatures between 60 and 80°        C., followed by cooling down to a temperature range of 35 to 45°        C.    -   2.3. Oil Solution Preparation    -   2.3.1. Mixture of vegetable oil with one of the following        compounds: Tween 80 0.2% (v□/□v) and/or a protective agent, as        for a non limiting example, laurel sodium sulphate 0.5% (v/v).    -   2.4. Capsule Preparation    -   2.4.1. Mixture of cellular suspension 1 to 20% (v/v) (see 2.1.)        with the polymer solution 1 to 5% (w/v) (see 2.2.).    -   2.4.2. Addition of the resulting mixture to 75 to 98% of the        prepared oil solution (see 2.3.). The obtained solution is        homogenised forming a water-in-oil emulsion.    -   2.4.3. The capsule formation occurs after the addition of a        solution of KCl 10 mM, for example, to the mixture at a        temperature range of 4 to 8° C.    -   2.5. Pre-fermentation process operation conditions

After attainment of the capsules with microorganisms for utilization ina fluidized bed reactor, the process of pre-fermentation is performed ata temperature between 20° C. to 52° C., during 4 to 8 hours, understerile and anaerobic conditions (circulating nitrogen flux), resultingin a fermented matrix. This suspension is drained into the reactor wherethe incorporation of the remaining food ingredients occurs.

3. Microorganisms Encapsulation Process by Emulsion and/or Spray-Drying

The microorganisms' encapsulation is done using the encapsulationtechniques:

-   -   3.1. Emulsion, as described in point 2;    -   3.2. Spray-drying:    -   3.2.1. Preparation of a cellular suspension with polymers (see        points 2.2; 2.2.1; and 2.4.1);    -   3.2.2. Drying of 250 ml of the previous mixture under the        constant conditions of inlet and outlet temperatures of        150-175° C. and 50-85° C., respectively;    -   3.2.3. Addition of the resulting powder into the pre-fermented        oat suspension (point 2.5) in a proportion of 2-5% (w/v), in a        way to guarantee 10⁸-10¹⁰ CFU in the matrix, per 100 g or 100        mL.

4. Food Ingredients Incorporation

Addition of ingredients to the matrix obtained in the previous process(point 3.2.3.), having as an example inulin, at a concentration rangebetween 1-3%, maintaining, as a non limitative example sea-salt, amongothers.

5. Presentation Forms of the Matrix

The pre-fermented symbiotic matrix based on an oat suspension withencapsulated probiotics can be presented either in a fresh form,lyophilized and/or frozen. The fresh matrix can be further presentedeither in gel or extruded form.

1. A pre-fermented symbiotic matrix comprising a suspension of cerealswith encapsulated microorganisms including at least one of the followingcompounds: free and/or encapsulated prebiotics; free microorganisms;other food ingredients.
 2. A pre-fermented symbiotic matrix according toclaim 1, wherein the cereals include flakes, flour or bran.
 3. Apre-fermented symbiotic matrix according to claim 2, wherein the cerealsinclude oats.
 4. A pre-fermented symbiotic matrix according to claim 2,wherein the cereals are combined with one or more other cereals and/orlegumes usually applied in food industry.
 5. A pre-fermented symbioticmatrix according to claim 3, wherein the cereals include oats combinedwith cereals and/or legumes, including barley and soy.
 6. Apre-fermented symbiotic matrix according to claim 1, wherein a prebioticsource includes β-glucan soluble fibres in biologically activequantities.
 7. A pre-fermented symbiotic matrix according to claim 6wherein β-glucan soluble fibres are extracted from cereals and/orlegumes.
 8. (canceled)
 9. A pre-fermented symbiotic matrix according toclaim 7, having a minimum of 0.75% (w/w) of β-glucan soluble fibres. 10.A pre-fermented symbiotic matrix according to claim 6, furthercomprising other prebiotic compounds including inulin,fructooligossacharides (FOS) and chitosans.
 11. A pre-fermentedsymbiotic matrix, according to claim 1, including integration of otherfood ingredients, that apart from the prebiotic function can still allowfor other functions, such as, organoleptic functions such as sweeteners,flavours and/or fruit pulp and texture such as enzymes.
 12. Apre-fermented symbiotic matrix, according to claim 1, including extraantioxidant sources, fatty acids such as omega 3, omega 6 and itsderivatives, vitamins and minerals beyond the already existent incereals and/or legumes, in a free or encapsulated form.
 13. Apre-fermented symbiotic matrix, according to claim 1, wherein themicroorganisms includes “GRAS”, probiotic and non-probiotic and whereinan encapsulate includes at least one of proteins, polysaccharides,lipids and hydrocolloids.
 14. A pre-fermented symbiotic matrix,according to claim 13, including inoculated microorganisms in a quantitynot inferior to 10⁸-10¹⁰ CFU/g, ensuring that the final product, whenconsumed, contains between 10⁶-10⁸ CFU/g.
 15. A pre-fermented symbioticmatrix, according to claim 1, wherein the matrix is in a fresh,lypophilised and/or frozen form.
 16. A pre-fermented symbiotic matrix,according to claim 15, wherein the fresh matrix includes a gel orextruded form.
 17. A process of obtaining a pre-fermented prebioticmatrix according to claim 1, comprising: a pre-fermentation includingplacing a cereal suspension in a reactor with immobilized microbialcells in macrocapsules, coated by proteins, polysaccharides, lipids andhydrocolloids; a separation wherein the microorganisms are separatedfrom the matrix; an encapsulation, including drying, atomization,emulsion or coacervation; and incorporating food ingredients. 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. (canceled)
 24. (canceled)
 25. The pre-fermented matrix according toclaim 12, wherein the microorganisms include the Bifidobacterium andLactobacillus genera.